The temperature dependence of Langmuir monolayers of normal and cancerous human cervical tissues and their organic phases between temperatures of 37 and 45 °C was evaluated. Analysis of the surface pressure-area isotherms revealed significantly different increase in fluidity of the cancerous cervical tissue monolayer at 42 °C as opposed to the normal cervical tissue monolayers (p < 0.05). Similarly, in the case of cervical cancerous organic phase monolayers significant increase of fluidity was observed at 40 °C whereas no such change was observed in the normal cervical organic phase monolayers. The effect of temperature was found to be different in cancerous and normal cervical tissues and this may be due to the different lipid profiles in them. Cancerous cervical tissues had 1.8-fold higher total lipids as compared to the normals. Similarly, the PC, PE, PI, PG, SM and PS levels in cancerous cervical tissues were 3.6, 2.0, 2.3, 4.7, 1.7 and 2.2 times higher than those of normal cervical tissues, respectively. Significant cancer-normal difference in minimum surface tension and hysteresis area was found at all temperatures studied for both tissue homogenates and organic phases. For example, cancerous tissue homogenates showed minimum surface tensions of 51.9 ± 4.6, 54.4 ± 5.9, 57.6 ± 6.0 and 51.9 ± 5.6 mN/m at temperatures 37, 40, 42 and 45 °C whereas the corresponding values for normal cervical tissue homogenates were 39.3 ± 3.6, 39.2 ± 3.7, 39.2 ± 3.8 and 39.1 ± 3.6, respectively. The fluidity change at hyperthermic range of temperature can be correlated to the increased efficiency of drug on combination therapy with hyperthermia. These results may have implications in manipulating the fluidity of cervical cancer tissue membranes for better permeability thereby leading to better therapeutic strategies for cervical cancer.